A Novel System to Diagnose Cutaneous Adverse Drug Reactions Employing the Cellscan—Comparison with Histamine Releasing Test and Inf-γ Releasing Test

Background: There are several mechanisms to describe allergic drug reactions yet the methods to diagnose them are limited

Acceleration of Autoimmunity by Organochlorine Pesticides in (NZB × NZW)F(1) Mice

Systemic lupus erythematosus (SLE) is an autoimmune disorder that affects women more frequently than men. In the (NZB × NZW)F(1) mouse, a murine SLE model, the presence or absence of estrogen markedly influences the rate of progression of disease. Three organochlorine pesticides with estrogenic effects were administered chronically to ovariectomized female (NZB × NZW)F(1) mice, and we measured the time to development of renal disease, the principal clinical manifestation of lupus in this model. Treatment with chlordecone, methoxychlor, or o,p′-dichlorodiphenyl-trichloroethane (o,p′-DDT) significantly decreased the time to onset of renal impairment, as did treatment with 17β-estradiol used as a positive control. In an expanded study of chlordecone, we found a dose-related early appearance of elevated anti–double-strand DNA autoantibody titers that corresponded with subsequent development of glomerulonephritis. Immunohistofluorescence confirmed early deposition of immune complexes in kidneys of mice treated with chlordecone. These observations are consistent with an effect of these organochlorine pesticides to accelerate the natural course of SLE in the (NZB × NZW)F(1) mouse. Although we originally hypothesized that the effect on progression of autoimmunity was due to estrogenic properties of the pesticides, autoimmune effects and estrogenicity, assessed through measurement of uterine hypertrophy, were not well correlated. This may indicate that uterine hypertrophy is a poor indicator of comparative estrogenic effects of organochlorine pesticides on the immune system, or that the pesticides are influencing autoimmunity through a mode of action unrelated to their estrogenicity

Application of a Static Fluorescence-based Cytometer (the CellScan) in Basic Cytometric Studies, Clinical Pharmacology, Oncology and Clinical Immunology

The CellScan apparatus is a laser scanning cytometer enabling repetitive fluorescence intensity (FI) and polarization (FP) measurements in living cells, as a means of monitoring lymphocyte activation. The CellScan may serve as a tool for diagnosis of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) as well as other autoimmune diseases by monitoring FP changes in peripheral blood lymphocytes (PBLs) following exposure to autoantigenic stimuli. Changes in FI and FP in atherosclerotic patients' PBLs following exposure to various stimuli have established the role of the immune system in atherosclerotic disease. The CellScan has been evaluated as a diagnostic tool for drug-allergy, based on FP reduction in PBLs following incubation with allergenic drugs. FI and FP changes in cancer cells have been found to be well correlated with the cytotoxic effect of anti-neoplastic drugs. In conclusion, the CellScan has a variety of applications in cell biology, immunology, cancer research and clinical pharmacology

Stimulation of tumour growth by wound-derived growth factors

The goal of this work was to determine the molecular basis for the induction of tumour vascularization and progression by injury. Magnetic resonance imaging (MRI) studies demonstrated that administration of wound fluid derived from cutaneous injuries in pigs reduced the lag for vascularization and initiation of growth of C6 glioma spheroids, implanted in nude mice, and accelerated tumour doubling time. The former effect can be attributed to the angiogenic capacity of wound fluid as detected in vivo by MRI, and in vitro in promoting endothelial cell proliferation. The latter effect, namely the induced rate of tumour growth, is consistent with the angiogenic activity of wound fluid as well as with the finding that wound fluid was directly mitogenic to the tumour cells, and accelerated growth of C6 glioma in spheroid culture. Of the multiple growth factors present in wound fluid, two key factors, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) and platelet-derived growth factor (PDGF), were identified as the dominant mitogens for C6 glioma, and inhibition of their activity using specific neutralizing antibodies suppressed the mitogenic effect of wound fluid on DNA synthesis in C6 glioma. This study suggests that the stimulatory effect of injury on tumour progression can possibly be attenuated by therapeutic targeting directed against a limited number of specific growth factors. © 1999 Cancer Research Campaig

Viral Superantigen Drives Extrafollicular and Follicular B Cell Differentiation Leading to Virus-specific Antibody Production

Mouse mammary tumor virus (MMTV[SW]) encodes a superantigen expressed by infected B cells. It evokes an antibody response specific for viral envelope protein, indicating selective activation of antigen-specific B cells. The response to MMTV(SW) in draining lymph nodes was compared with the response to haptenated chicken gamma globulin (NP-CGG) using flow cytometry and immunohistology. T cell priming occurs in both responses, with T cells proliferating in association with interdigitating dendritic cells in the T zone. T cell proliferation continues in the presence of B cells in the outer T zone, and B blasts then undergo exponential growth and differentiation into plasma cells in the medullary cords. Germinal centers develop in both responses, but those induced by MMTV(SW) appear later and are smaller. Most T cells activated in the T zone and germinal centers in the MMTV(SW) response are superantigen specific and these persist for weeks in lymph nodes draining the site MMTV(SW) injection; this contrasts with the selective loss of superantigen-specific T cells from other secondary lymphoid tissues. The results indicate that this viral superantigen, when expressed by professional antigen-presenting cells, drives extrafollicular and follicular B cell differentiation leading to virus-specific antibody production

Internal branding in universities and the lessons learnt from the past: the significance of employee brand support and transformational leadership

The paper reviews the literature on the concept of internal branding and its effects in the service sector in general, as well as in UK universities. In addition, the concept of employee brand support is reviewed, discussing the influence of leadership characteristics on internal branding in universities. Employee brand support is a crucial element to successful internal branding activities. A great concern is that the knowledge of internal branding and employee brand support in higher education institutions is based on knowledge imported from the business sector, and more practices should thus be adapted and implemented. It is found that transformation leadership, despite being little researched in the internal branding context, remains the pivotal element to the successful implementation of internal branding, as transformational leadership provides space for intellectual stimulation and consideration of individuals that are present at universities. Potential future research areas for internal branding studies are identified and discussed

Utilizing mitochondrial events as biomarkers for imaging apoptosis

Cells undergoing apoptosis show a plethora of time-dependent changes. The available tools for imaging apoptosis in live cells rely either on the detection of the activity of caspases, or on the visualization of exposure of phosphatidyl serine in the outer leaflet of the cell membrane. We report here a novel method for the detection of mitochondrial events during apoptosis, namely translocation of Bax to mitochondria and release of cytochrome c (Cyt c) using bimolecular fluorescence complementation. Expression of split yellow fluorescent protein (YFP) fragments fused to Bax and Cyt c, resulted in robust induction of YFP fluorescence at the mitochondria of apoptotic cells with very low background. In vivo expression of split YFP protein fragments in liver hepatocytes and intra-vital imaging of subcutaneous tumor showed elevated YFP fluorescence upon apoptosis induction. Thus, YFP complementation could be applied for high-throughput screening and in vivo molecular imaging of mitochondrial events during apoptosis

Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level

Repetitive Pertussis Toxin Promotes Development of Regulatory T Cells and Prevents Central Nervous System Autoimmune Disease

Bacterial and viral infections have long been implicated in pathogenesis and progression of multiple sclerosis (MS). Incidence and severity of its animal model experimental autoimmune encephalomyelitis (EAE) can be enhanced by concomitant administration of pertussis toxin (PTx), the major virulence factor of Bordetella pertussis. Its adjuvant effect at the time of immunization with myelin antigen is attributed to an unspecific activation and facilitated migration of immune cells across the blood brain barrier into the central nervous system (CNS). In order to evaluate whether recurring exposure to bacterial antigen may have a differential effect on development of CNS autoimmunity, we repetitively administered PTx prior to immunization. Mice weekly injected with PTx were largely protected from subsequent EAE induction which was reflected by a decreased proliferation and pro-inflammatory differentiation of myelin-reactive T cells. Splenocytes isolated from EAE-resistant mice predominantly produced IL-10 upon re-stimulation with PTx, while non-specific immune responses were unchanged. Longitudinal analyses revealed that repetitive exposure of mice to PTx gradually elevated serum levels for TGF-β and IL-10 which was associated with an expansion of peripheral CD4+CD25+FoxP3+ regulatory T cells (Treg). Increased frequency of Treg persisted upon immunization and thereafter. Collectively, these data suggest a scenario in which repetitive PTx treatment protects mice from development of CNS autoimmune disease through upregulation of regulatory cytokines and expansion of CD4+CD25+FoxP3+ Treg. Besides its therapeutic implication, this finding suggests that encounter of the immune system with microbial products may not only be part of CNS autoimmune disease pathogenesis but also of its regulation